Corn with increased yield and nitrogen utilization efficiency

ABSTRACT

The subject invention relates in part to the use of insect-protected corn to modify fertility recommendations for given yield targets on any transgenic corn type.

BACKGROUND OF THE INVENTION

Current fertility recommendations for corn were developed over a longperiod of time with traditional corn susceptible to insect attack, orwith corn protected from insects via application of chemicalinsecticides. Subject to the price of chemical fertilizers, typicalcurrent practice is for farmers to over-saturate their fields withfertilizers to a lesser or greater degree.

FIELD OF THE INVENTION

This invention is in the field of genetically engineered plants havingimproved yield.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns the surprising discovery that transgeniccorn that produces Bacillus thuringiensis (Bt) insecticidal toxins toprovide in-plant protection against feeding damage by above-ground andbelow-ground insect pests exhibits desirable agronomic characteristicsapart from the protection against insect feeding damage.

The subject invention relates in part to the use of insect-protectedtransgenic corn to modify fertility recommendations for a given yieldtarget. Specifically, it is found that transgenic corn commerciallyadopted as HERCULEX-XTRA; thus containing genes that encode Cry34Ab1,Cry35Ab1, and Cry1F insecticidal proteins, produces increased grainyield, as measured by kernel weight and number, when grown in fieldconditions and compared to isogenic control populations. It isadditionally found that these enhanced yields are obtained with afurther advantage of increased efficiency of nitrogen fertilizerutilization.

The present invention also includes methods of modulating nitrogenutilization efficiency (NUE) in a plant cell, comprising: (a)introducing into a plant cell a recombinant expression cassettecomprising a Cry protein operably linked to a promoter that drivesexpression in a plant; and (b) culturing the plant cell under plant cellgrowing conditions; wherein the nitrogen uptake in the plant cell ismodulated.

Other methods include increasing yield in a plant, wherein such methodscomprise the steps of: (a) introducing into a plant cell a constructcomprising a Cry protein operably linked to a promoter functional in aplant cell, so as to yield transformed plant cells; and, (b)regenerating a transgenic plant from said transformed plant cell,wherein said Cry protein is expressed in the cells of said transgenicplant at levels sufficient to increase yield in said transgenic plant;wherein increased yield comprises enhanced root growth, increased seedsize, increased seed weight, seed with increased embryo size, increasedleaf size, increased seedling vigor, enhanced silk emergence, increasedear size, nitrogen utilization or chlorophyll content.

Seeds and plants made from these methods may also be included.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1: cry34 plant-optimized gene sequence

SEQ ID NO:2: cry35 plant-optimized gene sequence

SEQ ID NO:3: truncated cry1F sequence encoding core toxin

SEQ ID NO:4: truncated/core-toxin Cry1F protein sequence

SEQ ID NO:5: native/full-length cry1F sequence

SEQ ID NO:6: native/full-length Cry1F protein sequence

SEQ ID NO:7: native cry34 sequence

SEQ ID NO:8: Cry34 protein sequence

SEQ ID NO:9: native cry35 sequence

SEQ ID NO:10: Cry35 protein sequence

DETAILED DISCLOSURE OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Langenheim and Thimann,(1982) Botany: Plant Biology and Its Relation to Human Affairs, JohnWiley; Cell Culture and Somatic Cell Genetics of Plants, vol. 1, Vasil,ed. (1984); Stanier, et al., (1986) The Microbial World, 5.sup.th ed.,Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant PathologyMethods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: ALaboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985);Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization,Hames and Higgins, eds. (1984); and the series Methods in Enzymology,Colowick and Kaplan, eds, Academic Press, Inc., San Diego, Calif.

As used herein “operably linked” includes reference to a functionallinkage between a first sequence, such as a promoter, and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame. As used herein, theterm “plant” includes reference to whole plants, plant organs (e.g.,leaves, stems, roots, etc.), seeds and plant cells and progeny of same.Plant cell, as used herein includes, without limitation, seeds,suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants, which can be used in the methods ofthe invention, is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants including species from the genera: Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.Plants of the invention include, but are not limited to, rice, wheat,peanut, sugarcane, sorghum, corn, cotton, soybean, vegetable,ornamental, conifer, alfalfa, spinach, tobacco, tomato, potato,sunflower, canola, barley or millet Brassica sp., safflower, sweetpotato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea,banana, palm, avocado, fig, guava, mango, olive, papaya, cashew,macadamia, almond, sugar beet, sugarcane, buckwheat, triticale, spelt,linseed, sugar cane, oil seed rape, canola, cress, Arabidopsis,cabbages, soya, pea, beans, eggplant, bell pepper, Tagetes, lettuce,Calendula, melon, pumpkin, squash and zucchini or oat plant. Aparticularly preferred plant is Zea mays.

As used herein, “yield” may include reference to bushels per acre of agrain crop at harvest, as adjusted for grain moisture (15% typically formaize, for example), and the volume of biomass generated (for foragecrops such as alfalfa, and plant root size for multiple crops). Grainmoisture is measured in the grain at harvest. The adjusted test weightof grain is determined to be the weight in pounds per bushel, adjustedfor grain moisture level at harvest. Biomass is measured as the weightof harvestable plant material generated.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples are promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, seeds, fibres, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as “tissuepreferred.” A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “regulatable” promoter is apromoter, which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light. Anothertype of promoter is a developmentally regulated promoter, for example, apromoter that drives expression during pollen development. Tissuepreferred, cell type specific, developmentally regulated, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter, which is active under mostenvironmental conditions, for example, the ubiquitin gene promoter Ubil.

Transgenic corn varieties with insect protection traits have beenavailable for several years. Multiple events are available for controlof above-ground and below-ground feeding insects. These events aretransformed with genes that produce Bt proteins and are marketed undernames such as HERCULEX, YIELDGARD and AGRISURE. They are widelyrecognized to be efficacious against insect damage and are recommendedfor planting by University field extension services, and by theirmanufacturers, in locations where insect damage is expected to adverselyaffect corn yields. The growing recommendations (e.g. fertilizerapplication rates) for all of these insect protected corn varieties areidentical to those recommended for nonprotected corn varieties.

While insect-protected corn varieties such as HERCULEX-XTRA arecurrently planted in many corn growing regions globally, farming methodsof growing them with reduced fertilizer recommendations were notpreviously taught or suggested and are a subject of the presentinvention. Fertilization rates, ranges, and amounts as describedelsewhere herein and as specifically exemplified in the Examples can beused to define methods of the subject invention. Various units and ratescan be used to express such rates and/or ranges—some of which are usedin the Examples (e.g., pounds of nitrogen fertilizer per acre).

Nitrogen fertilizer inputs are the third most costly input (behind landand seed) in corn production, and the cost may vary greatly depending onthe price of natural gas required to produce nitrogen fertilizer.Nitrogen use efficiency (NUE) represents an important target for maize(corn) breeding programs. Previous research demonstrates that geneticvariability exists for NUE and its components, N uptake efficiency(NUpE) and N utilization efficiency (NUtE). Thus, widespread adoption ofthe subject invention, having a result of less natural gas devoted tofertilizer production, can reduce costs for farmers as well as forconsumers who use natural gas for heating and cooking.

Grain yield in corn is largely affected by the ability of the plant totake up nitrogen (N) from the soil and utilize it for growth andreproduction. Plant responses to nitrogen fertilizer are observed acrossa wide range of application rates. The subject invention demonstratesthat grain yield and its components, kernel weight and number, areincreased across a range of N application rates by the presence of thetransgenic insecticidal proteins Cry34Ab1, Cry35Ab1 and Cry1F. Theseenhancements are accompanied by prolonged stay-green after flowering,and they correlate with increased N uptake efficiency before and afterflowering. Further, it is surprisingly seen that the plants of thesubject invention have increased grain yield per unit of plant nitrogen,both within and between N application rates. The effect is not seen withnontransgenic isogenic lines protected from insect attack by applicationof chemical insecticides, thus demonstrating that these benefits may bein addition to the root protection afforded by the transgenic insectcontrol.

The subject invention stems in part from our observation that yields ofgenetically similar corn lines, with and without the insect resistancetraits, unexpectedly respond differently to inputs, particularlynitrogen fertilizer. The insect-protected plants have less root damagefrom below-ground insect feeding. In addition, when damaged by rootfeeding insects such as corn root worm, the damaged plants regrowquicker and produce a larger root mass and have improved overall planthealth. This combination of factors allows the rate of nitrogen andother fertilizers to be reduced to obtain the same amount of yield as isobtained with non insect-protected corn lines grown with higherfertility amounts.

The present invention can comprise transgenic plants that accumulate theinsecticidal proteins Cry34Ab1 and Cry35Ab1 as well as CryF1. Theseproteins protect the root system of maize from damage from corn rootwormfeeding and facilitate improved N uptake and utilization, therebyproviding increased grain yield. Fertilizer application rates can bedetermined from the subject disclosure. Exemplary application rates,included within the subject invention, are demonstrated. Some preferredembodiments are also further specified in the Claims. Additionally,Herculex can be used in other crop species such as canola, wheat, rice,barley and other non-legume crops.

The present invention also includes methods of increasing nitrogenuptake efficiency in a plant by administering to a plant an expressioncassette containing at least one Bacillus thuringiensisinsect-resistance gene which functions to improve expression of at leastone insecticidal portion of a protein or amino acid sequence variantthereof from a nucleic acid coding sequence in a plant cell. Such methodcan further increase in the yield of the plant. The increase in yieldcan include an increase in the kernel number per plant and/or anincrease in the kernel mass per plant. Nitrogen utilization can also bemodulated. Additionally, the increase in nitrogen can occur during theflowering and grain filling periods of development.

The Bacillus thuringiensis insect-resistance gene used can include a Cryprotein. The insect-resistance gene can be selected from the groupconsisting of, e.g., a cry34 gene, a cry35 gene, a cry1F gene, and acry3A gene. The Bacillus thuringiensis insect-resistance gene cancomprise, e.g., Cry34Ab, Cry35Ab, and/or Cry1F. Sequences of therelevant proteins and genes of HERCULEX products are readilydeterminable. Unless otherwise indicated herein, the Cry1F protein andgene are as described in U.S. Pat. No. 6,218,188 (preferably thetruncated, plant-optimized version described therein), and the Cry34/35genes and proteins are as described in U.S. Pat. No. 6,340,593(preferably the 149B1 genes and proteins). Other genes and proteins thatcan be used according to the subject invention are also known in theart. See e.g. U.S. Pat. Nos. 7,179,965; 7,524,810; 7,939,651; 6,893,872;and 6,900,371. The Crickmore et al. website of the official Bacillusthuringiensis nomenclature committee is also well-known in the art andprovides links to many, publically available Cry protein and genesequences. Truncated and/or core-toxin fragments of Cry1F, for example,can be used, as is known in the art. Variants thereof are included. Theofficial nomenclature committee uses boundaries of at least 45% identity(e.g. Cry1F), 78% identity (e.g. Cry1Fa), and 95% protein sequenceidentity (Cry1Fa1) as primary, secondary, and tertiary ranks,respectively. Such boundaries, as well as 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,and/or 99% sequence identity (with an exemplified or suggested proteinor gene sequence) can be used according to the subject invention in someembodiments.

Either monocotyledonous plants or dicotyledonous plants may be used inthe present invention. The present invention also includes method ofgrowing transgenic corn plants having an increased yield by using areduced amount of fertilizer, wherein the transgenic corn is insectresistant due to expression of an insect-resistance gene, and whereinthe reduced amount of fertilizer is relative to fertilizer recommendedfor use on non-transgenic corn, wherein said non-transgenic corn isoptionally protected by granular chemical insecticide to controlrootworms. The increase in year can include an increase in the kernelnumber per plant or can include an increase in the kernel mass perplant. Such transgenic corn plants can yield corn comparable to cornyield from said non-transgenic corn grown using said recommended amountsof fertilizer. The fertilizer used can be a nitrogenous fertilizer. Thenitrogenous fertilizer can be applied at any rate of less than 150pounds per acre. Alternatively the nitrogenous fertilizer can be appliedat a rate of less than 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130,140 pounds per acre. In general, it may be preferable to have somenitrogenous fertilizer added to a crop. The nitrogenous fertilizer canbe applied to a field after planting said corn plants in the field butprior to emergence.

The present invention also includes methods of increasing yield ofmonocotyledonous plants or dicotyledonous plants due to nitrogenutilization.

Likewise, by means of the present invention, other agronomic genes canbe expressed in plants of the present invention. More particularly,plants can be genetically engineered to express various phenotypes ofagronomic interest. Exemplary genes implicated in this regard include,but are not limited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

-   -   A. Plant disease resistance genes. Plant defenses are often        activated by specific interaction between the product of a        disease resistance gene (R) in the plant and the product of a        corresponding avirulence (Avr) gene in the pathogen. A plant        variety can be transformed with cloned resistance genes to        engineer plants that are resistant to specific pathogen strains.        See, for example, Jones et al., Science 266:789 (1994) (cloning        of the tomato Cf-9 gene for resistance to Cladosporium fulvum);        Martin et al., Science 262:1432 (1993) (tomato Pto gene for        resistance to Pseudomonas syringae pv. tomato encodes a protein        kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2        gene for resistance to Pseudomonas syringae).    -   B. A gene conferring resistance to a pest, such as soybean cyst        nematode. See e.g., PCT Application WO 96/30517; PCT Application        WO 93/19181.    -   C. A Bacillus thuringiensis protein, a derivative thereof or a        synthetic polypeptide modeled thereon. See, for example, Geiser        et al., Gene 48:109 (1986), who disclose the cloning and        nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA        molecules encoding delta-endotoxin genes can be purchased from        American Type Culture Collection, Manassas, Va., for example,        under ATCC Accession Nos. 40098, 67136, 31995 and 31998.    -   D. A lectin. See, for example, the disclosure by Van Damme et        al., Plant Molec. Biol. 24:25 (1994), who disclose the        nucleotide sequences of several Clivia miniata mannose-binding        lectin genes.    -   E. A vitamin-binding protein such as avidin. See PCT application        US93/06487. The application teaches the use of avidin and avidin        homologues as larvicides against insect pests.    -   F. An enzyme inhibitor, for example, a protease or proteinase        inhibitor or an amylase inhibitor. See, for example, Abe et        al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of        rice cysteine proteinase inhibitor); Huub et al., Plant Molec.        Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding        tobacco proteinase inhibitor I); Sumitani et al., Biosci.        Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of        Streptomyces nitrosporeus alpha-amylase, inhibitor); and U.S.        Pat. No. 5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).    -   G. An insect-specific hormone or pheromone such as an        ecdysteroid or juvenile hormone, a variant thereof, a mimetic        based thereon, or an antagonist or agonist thereof. See, for        example, the disclosure by Hammock et al., Nature 344:458        (1990), of baculovirus expression of cloned juvenile hormone        esterase, an inactivator of juvenile hormone.    -   H. An insect-specific peptide or neuropeptide which, upon        expression, disrupts the physiology of the affected pest. For        example, see the disclosures of Regan, J. Biol. Chem.        269:9 (1994) (expression cloning yields DNA coding for insect        diuretic hormone receptor); and Pratt et al., Biochem. Biophys.        Res. Comm. 163:1243 (1989) (an allostatin is identified in        Diploptera puntata). See also U.S. Pat. No. 5,266,317 to        Tomalski et al., who disclose genes encoding insect-specific,        paralytic neurotoxins.    -   I. An insect-specific venom produced in nature by a snake, a        wasp, etc. For example, see Pang et al., Gene 116:165 (1992),        for disclosure of heterologous expression in plants of a gene        coding for a scorpion insectotoxic peptide.    -   J. An enzyme responsible for a hyperaccumulation of a        monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a        phenylpropanoid derivative or another non-protein molecule with        insecticidal activity.    -   K. An enzyme involved in the modification, including the        post-translational modification, of a biologically active        molecule; for example, a glycolytic enzyme, a proteolytic        enzyme, a lipolytic enzyme, a nuclease, a cyclase, a        transaminase, an esterase, a hydrolase, a phosphatase, a kinase,        a phosphorylase, a polymerase, an elastase, a chitinase and a        glucanase, whether natural or synthetic. See PCT application WO        93/02197 in the name of Scott et al., which discloses the        nucleotide sequence of a callase gene. DNA molecules which        contain chitinase-encoding sequences can be obtained, for        example, from the ATCC under Accession Nos. 39637 and 67152. See        also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993),        who teach the nucleotide sequence of a cDNA encoding tobacco        hornworm chitinase; and Kawalleck et al., Plant Molec. Biol.        21:673 (1993), who provide the nucleotide sequence of the        parsley ubi4-2 polyubiquitin gene.    -   L. A molecule that stimulates signal transduction. For example,        see the disclosure by Botella et al., Plant Molec. Biol. 24:757        (1994), of nucleotide sequences for mung bean calmodulin cDNA        clones; and Griess et al., Plant Physiol. 104:1467 (1994), who        provide the nucleotide sequence of a maize calmodulin cDNA        clone.    -   M. A hydrophobic moment peptide. See PCT application WO 95/16776        (disclosure of peptide derivatives of Tachyplesin which inhibit        fungal plant pathogens) and PCT application WO 95/18855 (teaches        synthetic antimicrobial peptides that confer disease        resistance).    -   N. A membrane permease, a channel former or a channel blocker.        For example, see the disclosure of Jaynes et al., Plant Sci.        89:43 (1993), of heterologous expression of a cecropin-.beta.,        lytic peptide analog to render transgenic tobacco plants        resistant to Pseudomonas solanacearum.    -   O. A viral-invasive protein or a complex toxin derived        therefrom. For example, the accumulation of viral coat proteins        in transformed plant cells imparts resistance to viral infection        and/or disease development effected by the virus from which the        coat protein gene is derived, as well as by related viruses. See        Beachy et al., Ann. rev. Phytopathol. 28:451 (1990). Coat        protein-mediated resistance has been conferred upon transformed        plants against alfalfa mosaic virus, cucumber mosaic virus,        tobacco streak virus, potato virus X, potato virus Y, tobacco        etch virus, tobacco rattle virus and tobacco mosaic virus. Id.    -   P. An insect-specific antibody or an immunotoxin derived        therefrom. Thus, an antibody targeted to a critical metabolic        function in the insect gut would inactivate an affected enzyme,        killing the insect. Cf. Taylor et al., Abstract #497, Seventh        Int'l Symposium on Molecular Plant-Microbe Interactions        (Edinburgh, Scotland) (1994) (enzymatic inactivation in        transgenic tobacco via production of single-chain antibody        fragments).    -   Q. A virus-specific antibody. See, for example, Tavladoraki et        al., Nature 366:469 (1993), who show that transgenic plants        expressing recombinant antibody genes are protected from virus        attack.    -   R. A developmental-arrestive protein produced in nature by a        pathogen or a parasite. Thus, fungal endo        alpha-1,4-D-polygalacturonases facilitate fungal colonization        and plant nutrient release by solubilizing plant cell wall        homo-alpha-1,4-D-galacturonase. See Lamb et al., Bio/Technology        10:1436 (1992). The cloning and characterization of a gene which        encodes a bean endopolygalacturonase-inhibiting protein is        described by Toubart et al., Plant J. 2:367 (1992).    -   S. A developmental-arrestive protein produced in nature by a        plant. For example, Logemann et al., Bio/Technology 10:305        (1992), have shown that transgenic plants expressing the barley        ribosome-inactivating gene have an increased resistance to        fungal disease.

2. Genes that Confer Resistance to an Herbicide:

-   A. An herbicide that inhibits the growing point or meristem, such as    an imidazolinone or a sulfonylurea. Exemplary genes in this category    code for mutant ALS and AHAS enzyme as described, for example, by    Lee et al., EMBO J. 7:1241 (1988); and Miki et al., Theon. Appl.    Genet. 80:449 (1990), respectively.-   B. Glyphosate (resistance conferred by, e.g., mutant    5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the    introduction of recombinant nucleic acids and/or various forms of in    vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate    acetyl transferase (GAT) genes, respectively), other phosphono    compounds such as glufosinate (phosphinothricin acetyl transferase    (PAT) genes from Streptomyces species, including Streptomyces    hygroscopicus and Streptomyces viridichromogenes), and pyridinoxy or    phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding    genes), See, for example, U.S. Pat. No. 4,940,835 to Shah, et al.    and U.S. Pat. No. 6,248,876 to Barry et. al., which disclose    nucleotide sequences of forms of EPSPs which can confer glyphosate    resistance to a plant. A DNA molecule encoding a mutant aroA gene    can be obtained under ATCC accession number 39256, and the    nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.    4,769,061 to Comai. European patent application No. 0 333 033 to    Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al.,    disclose nucleotide sequences of glutamine synthetase genes which    confer resistance to herbicides such as L-phosphinothricin. The    nucleotide sequence of a PAT gene is provided in European    application No. 0 242 246 to Leemans et al., DeGreef et al.,    Bio/Technology 7:61 (1989), describe the production of transgenic    plants that express chimeric bar genes coding for PAT activity.    Exemplary of genes conferring resistance to phenoxy proprionic acids    and cyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1,    Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theon. Appl.    Genet. 83:435 (1992). GAT genes capable of conferring glyphosate    resistance are described in WO 2005012515 to Castle et. al. Genes    conferring resistance to 2,4-D, fop and pyridyloxy auxin herbicides    are described in WO 2005107437 and U.S. patent application Ser. No.    11/587,893, both assigned to Dow AgroSciences LLC.-   C. An herbicide that inhibits photosynthesis, such as a triazine    (psbA and Is+ genes) or a benzonitrile (nitrilase gene). Przibila et    al., Plant Cell 3:169 (1991), describe the transformation of    Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide    sequences for nitrilase genes are disclosed in U.S. Pat. No.    4,810,648 to Stalker, and DNA molecules containing these genes are    available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning    and expression of DNA coding for a glutathione S-transferase is    described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

-   A. Modified fatty acid metabolism, for example, by transforming a    plant with an antisense gene of stearyl-ACP desaturase to increase    stearic acid content of the plant. See Knultzon et al., Proc. Natl.    Acad. Sci. U.S.A. 89:2624 (1992).-   B. Decreased phytate content-I) Introduction of a phytase-encoding    gene would enhance breakdown of phytate, adding more free phosphate    to the transformed plant. For example, see Van Hartingsveldt et al.,    Gene 127:87 (1993), for a disclosure of the nucleotide sequence of    an Aspergillus niger phytase gene. 2) A gene could be introduced    that reduced phytate content. In maize for example, this could be    accomplished by cloning and then reintroducing DNA associated with    the single allele which is responsible for maize mutants    characterized by low levels of phytic acid. See Raboy et al.,    Maydica 35:383 (1990).-   C. Modified carbohydrate composition effected, for example, by    transforming plants with a gene coding for an enzyme that alters the    branching pattern of starch. See Shiroza et al., J. Bacteol.    170:810 (1988) (nucleotide sequence of Streptococcus mutants    fructosyltransferase gene); Steinmetz et al., Mol. Gen. Genet.    20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase    gene); Pen et al., Bio/Technology 10:292 (1992) (production of    transgenic plants that express Bacillus lichenifonnis    alpha-amylase); Elliot et al., Plant Molec. Biol. 21:515 (1993)    (nucleotide sequences of tomato invertase genes); Sogaard et al., J.    Biol. Chem. 268:22480 (1993) (site-directed mutagenesis of barley    alpha-amylase gene); and Fisher et al., Plant Physiol.    102:1045 (1993) (maize endosperm starch branching enzyme II).-   D. Abiotic Stress Tolerance which includes resistance to    non-biological sources of stress conferred by traits such as    nitrogen utilization efficiency, altered nitrogen responsiveness,    drought resistance cold, and salt resistance. Genes that affect    abiotic stress resistance (including but not limited to flowering,    ear and seed development, enhancement of nitrogen utilization    efficiency, altered nitrogen responsiveness, drought resistance or    tolerance, cold resistance or tolerance, and salt resistance or    tolerance) and increased yield under stress.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Unless specifically indicated or implied, the terms “a”, “an”, and “the”signify “at least one” as used herein.

The present invention is explained in greater detail in the Examplesthat follow. These examples are intended as illustrative of theinvention and are not to be taken are limiting thereof. All percentagesare by weight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1

Corn hybrids derived from the IBMRIL population (Lee, M.; Sharopova, N.;Beavis, W. D.; Grant, D.; Katt, M.; Blair, D.; and Amel Hallauer, A.(2002) Expanding the genetic map of maize with the intermated B73×Mo17(IBM) population. Plant Molec. Biol. 48:453-461) were crossed toHERCULEX-XTRA and non-HERCULEX-XTRA isogenic testers. One hundred femaleinbreds were used in this experiment. These inbreds consisted of 99recombinant inbred lines from the intermated B73×Mo17 population(IBMRILs) and one Dow AgroSciences proprietary inbred (DASM7). Thegenotypes were consistent between 2008 and 2009 with the exception ofMO329 which was grown only in 2008 and MO379 which was grown only in2009. Furthermore, the hybrids formed from the parental inbred lines,B73 and Mo17, were only grown in 2009. The IBMRILs were chosen based onprevious data obtained in 2006 and 2007, and were selected to minimizeany confounding effects due to differences in maturity. Each of thefemale lines was crossed to both DASV8 and its near isogenic line,DASV8XT, which contains the HERCULEX-XTRA traits, to create a total of200 hybrids.

A split˜block design with three replications was used in which N rateand female parent were the whole plot treatments. Male parent wasincluded as a split˜plot within each N rate/female parent subplot. Plotswere planted on May 7, 2008 and May 21, 2009 on the University ofIllinois Cruse Farm in Champaign, Ill. All plots received an in˜furrowapplication of chlorpyrifos (Lorsban 15G) at a rate of 1.3 lbs a.i. peracre in 2008 and tefluthrin (Force 3G) at a rate of 0.099 lbs a.i. peracre in 2009. N was applied as ammonium sulfate ((NH₄)₂SO₄) in a diffuseband after emergence and incorporated between V2 and V3 plant growthstage. The N rates used in the study were 0 and 225 lbs per acre. Eachexperimental unit consisted of two rows spaced 2.5 feet apart. The rowswere 15 feet long in 2008 and 17.5 feet long in 2009. Plots were thinnedto an approximate density of 32,000 plants acre. At flowering, four(2008) or five (2009) representative plants were sampled and weighed. Ashredded aliquot was dried to constant weight and ground in a Wiley millto pass through a 20 mesh screen. Dried, ground stover samples wereanalyzed for total N concentration using a combustion technique (NA2000N˜Protein, Fisons Instruments, San Carlos, Calif.). A similar samplingapproach was used at the R6 plant growth stage except that the ears wereremoved, dried, and shelled to allow for calculation of per-plant grainweight at physiological maturity. At harvest, all plants within a singlerow of the two row plot were harvested. A subsample of the grain wasanalyzed for protein concentration using near-infrared transmittancespectroscopy (Foss 1241 NIT grain analyzer; FOSS, Eden Prairie, Minn.).Three-hundred kernels from each plot were counted using an electronicseed counter and weighed to obtain an estimate of individual kernelweight.

The phenotypic data were analyzed using the MIXED procedure of SAS.Nitrogen rate, female parent, and male parent were treated as fixedeffects while replication was considered random.

Genetic utilization (GU) is defined as grain weight (kg) per unit ofplant N (kg) under nonfertilized conditions, and has units of kg/kgplantN. Nitrogen Use Efficiency (NUE) was calculated as the ability toproduce grain (kg) per unit of added fertilizer N (kg) and has units ofkg/kgN. Nitrogen Uptake Efficiency (NUpE) was calculated as thedifference in plant nitrogen content at high and low N applicationlevels, divided by the difference in the applied levels of N. This is ameasure of the efficiency of fertilizer N uptake into the plant per unitof fertilizer N (kg) and has units of kg plantN/kgN. NitrogenUtilization Efficiency (NUtE) is defined as the grain weight (kg) perunit of N taken up by plant and has units of kg/kg plantN

The fields in which these hybrids were grown were treated with chemicalpesticides at rates recommended by the manufacturer to control cornrootworms. Thus control of root damage was not dependent upon efficacyof the Cry34Ab1 and Cry35Ab1 HERCULEX-XTRA genes. The data summarized inTable 1 show that when averaged across the 99 IBMRIL female parents, inboth 2008 and 2009, the hybrids with the HERCULEX-XTRA trait (DASV8XT)had significantly better Nitrogen Uptake Efficiency (NUpE) than did thehybrids which did not contain the HERCULEX-XTRA genes.

The values of Nitrogen Use Efficiency (NUE) and Nitrogen UtilizationEfficiency (NUtE) were found not to correlate with the presence/absenceof the HERCULEX-XTRA traits in 2008 and 2009 (Table 1)

TABLE 1 N use components of corn lines having the HERCULEX-XTRA traits(DASV8XT) compared to isogenic lines without the traits (DASV8) atChampaign, IL in 2008 and 2009, at two N application rates. N rate Male2008 2009 Lbs/acre parent GU^(a) NUE^(b) NUpE^(c) NUtE^(d) GU^(a)NUE^(b) NUpE^(c) NUtE^(d) 0 DASV8 49.5a 67.4a 0 DASV8XT 46.b 66.0a 225DASV8 12.9a 0.32a 40.7a 30.5a 0.56a 55.4a 225 DASV8XT 14.0b 0.35b 42.4a29.2b 0.60b 49.3b Means within a column followed by the same letter arenot significantly different at P ≦ 0.05. ^(a)Genetic Utilization (GU).Defined as grain weight (kg) per unit of plant N (kg) undernonfertilized conditions. Units are kg/kgplantN. ^(b)N Use Efficiency(NUE) Defined as ability to produce grain (kg) per unit of addedfertilizer N (kg). Units are kg/kgN. ^(c)N Uptake Efficiency (NUpE).Defined as efficiency of fertilizer N uptake into the plant (kg) perunit of fertilizer N (kg). Units are kgplantN/kgN. ^(d)N UtilizationEfficiency (NUtE). Defined as grain weight (kg) per unit of N taken upby plant (kg). Units are kg/kgplantN.

The increased Nitrogen Uptake Efficiency seen with plants having theHERCULEX-XTRA traits was reflected in increased plant N content at theR6 physiological state (Table 2) in both 2008 and 2009.

TABLE 2 Effect of N level and presence of the HERCULEX-XTRA trait on Ncontent at physiological maturity (R6) at Champaign, IL in 2008 and2009. 2008 2009 N rate N content N content lbs/acre Male parent gm/plantgm/plant 0 DASV8 0.7a 0.7a 0 DASV8XT 0.8b 0.8b 225 DASV8 1.8c 2.9c 225DASV8XT 1.9d 3.1d Means within a column followed by the same letter arenot significantly different at P ≦ 0.05.

Further, the HERCULEX-XTRA containing hybrids had significantly betteryield traits than did the hybrids which did not contain theHERCULEX-XTRA genes (Table 3). Yield was increased based on both anincrease in kernel number per plant and an increase in kernel massImproved yield correlated with higher N content at the R6 stage in bothyears of the study. The data Table 2 and Table 3 taken togetherdemonstrate that improved yield correlated with higher N content at theR6 stage. Thus, one skilled in the field of crop physiology will realizethat the HERCULEX-XTRA traits are effective in causing an increase in Nuptake during the flowering and grain filling periods on development.

TABLE 3 Grain yield and yield components of corn lines having theHERCULEX-XTRA traits (DASV8XT) compared to isogenic lines without thetraits (DASV8) at Champaign, IL in 2008 and 2009, at two N applicationrates. 2008 2009 Grain Kernel Kernel Grain Kernel Kernel N rate MaleRepro. yield weight Number Repro. yield weight Number Lbs/acre parentSuccess % Bu/acre mg/kernel Per plant Success % Bu/acre mg/kernel Perplant o DASV8 93a  69a 229a 218a 74a  73a 218a 323a o DASV8XT 95b  88b258b 240b 83b  85b 235b 315b 225 DASV8 99c 130c 216c 408c 98c 218c 264c605c 225 DASV8XT 99c 154d 251b 418d 99c 224d 275d 593d Means within acolumn followed by the same letter are not significantly different at P≦ 0.05.

Ma et al. (Ma, B. L.; Meloche, F.; and Wei, L: (2009) Agronomicassessment of Bt trait and seed or soil-applied insecticides on thecontrol of corn rootworm and yield. Field Crops Research. 111^(th)edition: 189-196) showed little to no yield effect of Cry3Bb1 events(active against corn rootworms) in a commercial hybrid compared with anisogenic non-transgenic hybrid treated with Force 3G insecticide tocontrol corn rootworms. Likewise, Vigna (Vigna, M. M. (2008) Comparisonof the effect of corn rootworm technology and seed-applied insecticide(clothianidin) to nitrogen status in corn. MA Thesis, Iowa StateUniversity, Ames) found no difference in yield or N content oftransgenic hybrids containing either Cry3Bb1 or Cry34Ab1+Cry35Ab1 (eventDAS59122-7) with isogenic non-transgenic controls treated with Poncho1250. Thus, the HERCULEX-XTRA transgenes may have broader effects thansimply limiting rootworm damage.

Increasing nitrogen uptake efficiency and yield by production ofHERCULEX-XTRA proteins in plants could have a dramatic effect onagriculture if also effective in crop species such as canola, wheat,rice, barley and other non-legume crops.

TABLE 4 Analysis of variance results for grain yield and N use traitsmeasured in 2008. Source of Variation Trait N rate Female N rate ×female Male parent N rate × male Female × male N rate × female × male R1whole shoot biomass 0.0051 0.0041 0.0163 <0.0001 0.0150 n.s.* n.s. R1whole shoot N content 0.0018 n.s. n.s. n.s. n.s. n.s. n.s. R6 grainweight 0.0004 <0.0001 n.s. <0.0001 0.0043 n.s. n.s. R6 whole shootbiomass 0.0054 <0.0001 n.s. 0.0424 n.s. n.s. n.s. R6 total N content0.0016 0.0083 n.s. <0.0001 n.s. n.s. n.s. Harvest index <0.0001 <0.00010.0318 n.s. n.s. 0.0386 n.s. Nitrogen harvest index <0.0001 0.0003 n.s.n.s. n.s. n.s. n.s. Grain protein concentration <0.0001 <0.0001 <0.00010.0188 0.0011 0.0948 n.s. Yield <0.0001 <0.0001 0.0524 <0.0001 0.05160.0325 n.s. Kernel weight 0.0004 <0.0001 <0.0001 <0.0001 0.0857 0.0005n.s. Kernel number 0.0010 <0.0001 n.s. <0.0001 0.0193 n.s. n.s.Reproductive success 0.0189 n.s. 0.0259 0.0164 0.0027 n.s. n.s. NUE —0.0279 — 0.0018 — n.s. — NUpE — n.s. — 0.0011 — n.s. — NUtE — 0.0385 —n.s. — n.s. — GU — 0.0004 — <0.0001 — n.s. — *Non-significant sourcevariation (n.s.).

TABLE 5 Analysis of variance results for grain yield and N use traitsmeasured in 2009. Source of Variation Trait N rate Female N rate ×female Male parent N rate × male Female × male N rate × female × male R1whole shoot biomass 0.0200 <0.0001 n.s.* <0.0001 n.s. 0.0153 n.s. R1whole shoot N content 0.0014 0.0117 0.0831 <0.0001 <0.0001 0.0905 n.s.R6 grain weight 0.0023 <0.0001 0.0001 0.0015 0.0017 0.0870 0.0489 R6whole shoot biomass 0.0012 <0.0001 0.0007 <0.0001 0.0598 0.0013 0.0178R6 total N content 0.0003 <0.0001 0.0001 <0.0001 0.0004 <0.0001 <0.0001Harvest index 0.0057 <0.0001 <0.0001 0.1091 <0.0001 n.s. n.s. Nitrogenharvest index 0.0513 0.0002 <0.0001 n.s. <0.0001 n.s. n.s. Grain proteinconcentration 0.0005 <0.0001 <0.0001 <0.0001 n.s. 0.0329 n.s. Yield0.0005 <0.0001 <0.0001 <0.0001 0.0007 0.0003 0.0440 Kernel weight 0.0009<0.0001 0.0015 <0.0001 <0.0001 <0.0001 n.s. Kernel number 0.0008 <0.00010.0016 <0.0001 n.s. 0.0024 n.s. Reproductive success 0.0016 <0.0001<0.0001 <0.0001 <0.0001 0.0060 0.0005 NUE — <0.0001 — 0.0003 — 0.0140 —NUpE — <0.0001 — <0.0001 — 0.0002 — NUtE — <0.0001 — <0.0001 — <0.0001 —GU — <0.0001 — 0.0735 — n.s. — *Non-significant source variation (n.s.).

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

We claim:
 1. A method of increasing nitrogen uptake efficiency in aplant comprising administering to a plant an expression cassettecontaining at least one Bacillus thuringiensis insect-resistance genewhich functions to improve expression of at least one insecticidalportion of a protein or amino acid sequence variant thereof from anucleic acid coding sequence in a plant cell.
 2. The method according toclaim 1 wherein said method further comprises an increase in the yieldof the plant.
 3. The method according to claim 2 wherein the increase inyield comprises an increase in the kernel number per plant and/or anincrease in the kernel mass per plant.
 4. The method according to claim1 further comprising culturing the plant under plant cell growingconditions wherein the nitrogen utilization is modulated.
 5. The methodaccording to claim 4 wherein said Bacillus thuringiensisinsect-resistance gene encodes a Cry protein.
 6. The method according toclaim 5 wherein said insect-resistance gene is selected from the groupconsisting of a cry34 gene, a cry35 gene, a cry1F gene, and a cry3Agene.
 7. The method according to claim 1 wherein the Bacillusthuringiensis insect-resistance gene comprises Cry34Ab1, Cry35Ab1, andCry1F.
 8. The method according to claim 1 wherein the increase innitrogen occurs during the flowering and grain filling periods ofdevelopment.
 9. The method according to claim 1 wherein the plant isselected from the group consisting of a monocotyledonous plant and adicotyledonous plant.
 10. The method according to claim 1 wherein theplant is a monocotyledonous plant.
 11. A method of growing transgeniccorn plants having an increased yield by using a reduced amount offertilizer, wherein the transgenic corn is insect resistant due toexpression of an insect-resistance gene, and wherein the reduced amountof fertilizer is relative to fertilizer recommended for use onnon-transgenic corn, wherein said non-transgenic corn is optionallyprotected by granular chemical insecticide to control rootworms.
 12. Themethod according to claim 11 wherein said transgenic corn plants yieldcorn comparable to corn yield from said non-transgenic corn grown usingsaid recommended amounts of fertilizer.
 13. The method according toclaim 11 wherein said fertilizer is a nitrogenous fertilizer.
 14. Themethod according to claim 11 wherein said transgenic plant comprises aBacillus thuringiensis insect-resistance gene.
 15. The method accordingto claim 14 wherein said Bacillus thuringiensis insect-resistance geneencodes a Cry protein.
 16. The method according to claim 14 wherein saidinsect-resistance gene is selected from the group consisting of a cry34gene, a cry35 gene, a cry1F gene, and a cry3A gene.
 17. The methodaccording to claim 14 wherein the Bacillus thuringiensisinsect-resistance gene comprises Cry34Ab1, Cry35Ab1, and Cry1F.
 18. Themethod according to claim 13 wherein said nitrogenous fertilizer isapplied at a rate of less than 150 pounds per acre.
 19. The methodaccording to claim 11 wherein said transgenic corn plants are grown in afield, and said fertilizer is nitrogenous fertilizer applied to saidfield after planting said corn plants in said field but prior toemergence.
 20. A method of modulating nitrogen utilization efficiency(NUE) in a plant cell, comprising: (a) introducing into a plant cell arecombinant expression cassette comprising a Cry protein operably linkedto a promoter that drives expression in a plant; and (b) culturing theplant cell under plant cell growing conditions; wherein the nitrogenuptake in the plant cell is modulated.
 21. A method for increasing yieldin a plant, said method comprising the steps of: (a) introducing into aplant cell a construct comprising a Cry protein operably linked to apromoter functional in a plant cell, so as to yield transformed plantcells; and, (b) regenerating a transgenic plant from said transformedplant cell, wherein said Cry protein is expressed in the cells of saidtransgenic plant at levels sufficient to increase yield in saidtransgenic plant; wherein increased yield comprises enhanced rootgrowth, increased seed size, increased seed weight, seed with increasedembryo size, increased leaf size, increased seedling vigor, enhancedsilk emergence, increased ear size, nitrogen utilization or chlorophyllcontent.
 22. Seed from the transgenic plant of claim 21.